CN101730537A - Have the pharmaceutical composition that comprises nardgrass of antifungal activity, method of acquisition nardgrass and uses thereof - Google Patents

Abstract

The present invention relates to the nardgrass extract and be used to prepare application, be used to obtain the method for described extract, and described extract is as the purposes that is used for the treatment of the active component of mycotic described compositions with anti-fungal activity medicaments compositions.The invention still further relates to the compositions that comprises plant extract, described plant extract derives from the aerial parts of the plant of the grass family family with antifungal activity.Preferably, described plant extract derives from the leaf of plant.More preferably, the plant of use is nardgrass (Cymbopogon nardus (L.) Rendle).

Description

Have the pharmaceutical composition that comprises nardgrass of antifungal activity, method of acquisition nardgrass and uses thereof

Invention field

The present invention relates to the nardgrass extract and be used to prepare application, be used to obtain the method for described extract, and described extract is as the purposes that is used for the treatment of the active component of mycotic described compositions with anti-fungal activity medicaments compositions.

The invention still further relates to the compositions that comprises plant extract, described plant extract derives from the aerial parts of the plant of the grass family family with antifungal activity.Preferably, described plant extract derives from the leaf of plant.More preferably, the plant of use is nardgrass (Cymbopogonnardus (L.) Rendle).

The invention still further relates to compositions and be used for the treatment of the purposes of mycosis (tinea unguium, dermatomycosis and vagina candidiasis), described compositions comprises the plant extract and/or the isolating material of the plant part that derives from the plant nardgrass.

Prior art

Dermatomycosis is to influence for example common disease of fingernail and toenail and hair of all groups' infringement skin and appurtenance.Although these diseases are not important relating to aspect the M ﹠ M, they influence patient's quality of life.Because communicable character, they have the important clinical consequence, especially are reflected in the aesthetic prejudice of self-respect, vanity and social discrimination aspect.Importantly will emphasize the seemingly global tendency of remarkable increase of the sickness rate of these diseases, these mycotic chronic disease character and treatment difficulty also are the factors that increases the weight of.

Traditionally, the stimulus object of dermatomycosis is a dermatophytes: the fungus that belongs to trichophyton (Trichophyton spp), Microsporon (Microsporum spp) and three genus of Epidermophyton (Epidermophytom floccosun).Yet the yeast of Candida, Piedraia and Geotrichum and the filamentous fungi of non-dermatophytes such as the mould genus of Fusarium, Scytalidium and broom have had making a definite diagnosis of certain frequency.

Tinea unguium (OM) is the common disease of infringement human finger's first and toenail and can influences everyone in indistinction ground.Because the infectiousness character of OM, OM has the important clinical consequence, especially is reflected in the aesthetic prejudice of self-respect, vanity and social discrimination aspect, is the major reason of going to a doctor or even losing employment.They have represented 20% of disease of nail, and are to be modal onychonosus on All Around The World.Most of author diagnoses dermatophytes as modal pathogen (80-90%), follows by yeast (5-17%), and be non-dermatophytes filamentous fungi (2-12%) at last.

Because fungus keeps being confined to the outermost layer of fingernail, some authors recommend topical therapeutic as first-selection.Topical therapeutic is applicable to local infection and almost not expansion, in invalid situation, must open the prescription of the medicine of whole body administration.Yet whole body therapeutic is more effective for chronic infection especially.

Although the undeniable progress aspect the exploitation of new antifungal agent, the treatment of tinea unguium are still a problem.

Another kind infection with originated from fungus of big dependency is vagina candidiasis (CVV), is called vaginal candidiasis again, is the mucous inflammation of reproduction of mainly damaging V﹠V.Modal pathogen be Candida albicans (a kind of yeast that has the two condition feature) and can 20% health with asymptomatic human female in find.Cardinal symptom as the CVV feature is to itch and pudendum-vagina erythema, the edema and the breach (Garcia in the white of cheese outward appearance and dense thick ejection, the calcination of urinating (disury), sexual relationship pain (disparunia), pudendum zone; Svidzinski, 2002).This diagnosis of infection can be by characteristic symptom, identify to determine (Daniel by vagina pH (be 4-4.5 normal range), outward appearance by secretions and abnormal smells from the patient with by the microbiological examination of yeast and mycelia; Robinson, 2005).

It is one of the women of child-bearing age's the main gynecological problem that influences that influences thousands of people in the whole world, and the sickness rate in recent years seemingly increases to some extent.According to estimates, the adult women near 75% at least fungoid pudendum-colpitic acute attack occurs in its adult phase, and 40-50% lives through (CVVR) that new outbreak and 5% becomes recurrent.

The situation of the 80-90% of CVV is because Candida albicans belongs to, and 10-20% is owing to be called as other species (Oidium tropicale (C.tropicalis), Candida glabrata (C.glabrata), Candida krusei (C.krusei), Candida parapsilosis (C.parapsilosis)) of non-Candida albicans (C.non-albicans).Yet, observed that the saccharomycetic cross frequence of non-Candida albicans increases to some extent in some populations.Main problem is that these other species tend to more tolerate this fact of antifungal usually.

In recent years, pathogen increases to some extent for the drug resistance of common therapeutic agent.At present, in the classic treatment method of tinea unguium, have medicine for example terbinafine and azole derivative can use, but cost is too high, has shifted that treatment is selected and success rate is limited.

CVV is modal diagnosis and increasing to some extent recent years in the gynecology everyday practice, yet its treatment is still that doctor and patient worry, and mainly is effect and recurrence to symptom.

The treatment of CVV is depended on and medicine, host and the obvious relevant variable of environmental factors.Available treatment comprises topical drug's (nistatine and miconazole) and oral drugs (mainly be azole derivative, for example fluconazol, itraconazole and ketoconazole (cetoconazol)).

Make progress to some extent aspect the new antifungal of CVV although closely be used for the treatment of in exploitation decades, it is still an important problem.Present available optimum selection is limited and is expensive treatment for the patient.

Carrying out countless research aspect the chemical compound that has biologic activity from the natural product searching.Carried out the development that many research evaluations derive from the antimicrobial acivity of the extract of medicinal plants and quintessence oil.Many plants have resistance to different pathogen, and this resistance may be relevant with the existence of the chemical compound of the inhibition fungus of natural generation.

The knowledge of having understood the medicinal plants that will use in passing at looking for of the treatment alternative of plant amedica therapy, optimized extraction, obtained being applicable to treatment and had the medicament forms that in treatment, obtains the required character renderd a service.Require similarly with these, still need the check analysis technology and make the plant extract standardization.

In the whole world, the Therapeutic Method of plant origin has huge popularize and popular, and in general, the Therapeutic Method of plant origin huge popularized and popular burden is so not heavy and be shown as replenishing of health care service.Seeking help from before other has more rodent medicine, they can be the first-selections of multiple disease.

Medicinal plants produces the various ingredients with countless performance, and it can suppress the growth of pathogen or kill them, and for this reason, they are considered to develop the optimum selection of new antimicrobial drug.

Nardgrass (Cymbopogon nardus (L.) Rendle) generically is called as Herba Cymbopogonis Citrari, it is the plant of grass family family, common in the torrid zone or subtropical climate, it appears at Ceylon and South India the earliest, and its quintessence oil is rich in oil of citronella, isopulegol and geraniol, usually as anthelmintic and disinfectant.

Nardgrass to the rejection activity of insecticide by extensively open, relevant with the existence of oil of citronella in the quintessence oil and isopulegol.Recently, proved the effect of the relevant nardgrass of-aletrine trans with D-to Aedes aegypti, when using the volatile oil of 0.1% concentration, insect mortality is near 88.9%.

The example that relates to the insecticidal activity of nardgrass in the prior art individually or in combination can find in Brazilian patent document PI9106328-0, it relates to the anthelmintic of spray or lotion form, comprise terpinol (turpeniol), oil of citronella, rodenol extract and geraniol, flat louse and the sting insect and the Triatoma insecticide (card Ge Shi insecticide (Chagas insects)) of Lyme disease propagated in the collaborative antagonism effectively of each composition.

Similarly, PI9610350-7 relates to the anthelmintic mixture of at least two kinds of selected components that comprise mentanodiol and oil of citronella, geraniol, terpinol and rhodinol (rodinol), is used to resist mosquito and flat louse.

In addition, the preliminary study of using the plant of grass family family to carry out is verified at the important bactericidal activities of various human infection funguses.

By the multidigit authors report quintessence oil of other species of nardgrass or Cymbopogon to the extracorporeal antifungal activity of interested fungus medically.

Yet this research does not have report to be used for the treatment of the proof of the purposes of tinea unguium, female vulva vaginal candidiasis and trichomoniasis about the existence of determining similar activity and plant amedica therapy compositions in the nardgrass extract and they.

The purposes of this extract can promote to obtain the process of plant amedica therapy medicine greatly, significantly reduces the cost of described product, and helps this active component is incorporated in the different medicament forms.

Therefore, derive from natural product (mainly being plant origin) biologic activity noval chemical compound look for that to become most interested target be because to the needs of the new drug of the multiple disease of effective antagonism.

Relevant with the anti-parasite activity of oil of citronella, the claimed plant amedica therapy product of PI0106192-5 based on citronella oil, it is briefly presenting big effectiveness aspect the interior and epizoa of treatment and the common attack of prevention cattle, sheep, goat and horse.

About antimicrobial acivity, also known PI9804814, it has described oral hygiene composition, and described compositions comprises and is selected from following antimicrobial: the extract of Cedrus deoclar (Roxb.) G. Don oil, chloramphenicol (cloranfenicol), Indian oil of verbena, citronella oil, Glycyrrhiza glabra L. (Glycyrrhizinglabra), band juice Fruit oil, Fructus Citri Limoniae

The oil of oil (Ocimumsp) and Herba Rosmarini Officinalis (Rosmarinus officinaLIs).

PI0106903-9 relates to the pharmaceutical composition that is used for the treatment of oral cavity and vaginal candida, water alcohol, pure and mild ethereal extract and/or the quintessence oil of aerial parts that comprises the lemon grass (Cymbopogon citratus) (Cymbopogon citratus stapf) (grass family) of 1-5 weight % is the form of the mixture of the different proportion of independent form or the different proportion mixture of himself or or sintetics (medicine, vitamin, salt, sugar etc.) natural with other.Pharmaceutical preparation can be used as tincture, Emulsion A/O and O/A, cream, gel, aerosol, paste, soap, shampoo and is used for the local similar products that use, and is used for the treatment of the infection that is caused by fungus and antibacterial (the particularly candidiasis of oral cavity and vagina).

PI0203521-9 by Candida and dermatophytes (has for example considered, Epidermophyton (Epidermophyton floccusum), Sabouraudites lanosus (Microsporum canis) and trichophyton purpureatum (trichophyton rubrum)) pharmaceutical composition that causes, it comprises the volatile oil of the Herba Cymbopogonis Citrari (Cymbopogon citratus) (lemon grass (Cymbopogon citratus) (lemon grass)) of active drug amount of science.In described compositions, volatile oil extracts from the leaf that is used for distillatory lemon grass (Cymbopogon citratus) by stripping, contains citral (60-80%v/v), myrcene (mircene) and geraniol as main component.

Patent JP7061918 has disclosed a kind of cosmetics, comprise and at least aly have strong antioxidant action and can keep skin not ftracture and have the extract of aura, described extract is selected from Caulis Miscanthis floriduli (Vetiveria zizanoides), Indian sarsaparilla (Hemidesmus indicus), nardgrass (Cymbopogon nardus), Fructus Piperis Longi (Piper longum), Java long fruit Fructus Piperis (Piper chaba), Herpestris monnies, Herba Cardiospermi microcarpi (Cardiospermumhalicacabum), Tinosooracordifolia (Tinospora cordifolia), glochild pod mountain Hirudo (Desmodium gangeticum), Huang Lan (Michelia champaca) and Melaleucaleucadendroncom.

Also know from prior art, extraction is known from the volatile oil of nardgrass to the active application of the rejection of insecticide, cleaning and disinfection product and about the application of Herba Cymbopogonis Citrari class (lemon grass (Cymbopogon citratus)), all is the antibiotic pharmacological activity about antagonism Candida (Candida spp) and dermatophytes (dermatofite fungi).Yet the neither one document once related in the past or the compositions of advising comprising plant nardgrass extract has to anti-dermatophyte (dermatofite fungi) with to the activity of anti-candida albicans (Candida albicans).

Therefore, prior art does not have description or advises the purposes of nardgrass extract as antifungal.

As seen, the treatment of tinea unguium, dermatomycosis and those vulvovaginal candidiasiss is still a problem, although obtaining undeniable progress aspect the exploitation of new antifungal agent.Therefore, use the product of the plant origin with fungicide performance can bring the big interests that are used to resist tinea unguium, female vulva vaginal candidiasis and dermatomycosis, this does not obtain research as yet.

Therefore, all be easy to generate and growing plants because nardgrass is a all regions in Brazil, do not have specific (special) requirements for cultivating, therefore, its extract can obtain with quite low production cost.In addition, the acquisition of its extract show good yield and it is incorporated in the pharmaceutical dosage form quite feasible, for the leaf that replaces this kind of plant and the industrialization of stem are provided convenience.Biological results of the present invention shows that high antifungal activity causes the death (antifungal) of test microorganism, and this is the significant advantage that is better than available most of antifungal on the market (its major part only is to suppress fungus).

Although report has compositions and medicine by obtaining from plant extract in the literature, do not mention or advise having the purposes of plant amedica therapy compositions of the pure and mild standardized extract that comprises nardgrass of antifungal activity and their extracting method.This extracting method, plant amedica therapy compositions and their purposes are described in this application and are claimed.

Summary of the invention

The present invention has predicted the pharmaceutical composition that is enough to be used in treating tinea unguium and female vulva vaginal candidiasis, and it has utilized the nardgrass water-alcohol extraction and the acceptable excipient of pharmacy of pharmacological activity amount.

The present invention relates to the water-alcohol extraction of standardized method, particularly nardgrass of the plant of the method that obtains water-alcohol extraction, dry extract and grass family family in addition.

The invention still further relates to have the antifungal activity that is used for tinea unguium compositions to anti-dermatophyte and oidiomycetic purposes, described compositions comprises the plant extract of aerial parts of the plant (more specifically for nardgrass) of grass family family.

The accompanying drawing summary

In order to show scope of the present invention better, the activity determined in the plant-nardgrass of using and the extract therefrom and the various illustrative figure of performance are provided in the present invention in advance, wherein:

The distribution of the minimum inhibitory concentration value (mg/ml) when-Fig. 1 illustrates the nardgrass extract to 100% inhibition index of dermatophytes and saccharomycetic separator.

-Fig. 2 illustrates the minimal fungicidal concentration (mg/ml) of can 100% killing the nardgrass extract of dermatophytes and saccharomycetic separator.

-Fig. 3 illustrates the method for analyzing the nardgrass antifungal activity.

Detailed Description Of The Invention

First aspect be the present invention relates to comprise the nardgrass water-alcohol extraction be used for the treatment of onychomycosis, dermatomycosis and oidiomycotic pharmaceutical composition.

Pharmaceutical composition of the present invention can comprise the nardgrass water-alcohol extraction that concentration is 5-40% (p/p).

More preferably, described pharmaceutical composition can comprise the nardgrass water-alcohol extraction that concentration is 10-30% (p/p).

Pharmaceutical excipient uses to be suitable for every kind of selected medicament forms of disease to be treated.

The liquid form of pharmaceutical composition uses the water-alcohol solvent of C1-C10 carbon atom, such as for example, and ethanol and dipropylene glycol.Dipropylene glycol can be that 0.5: 20, more preferably 1: 10 ratio is used with the dry extract with respect to nardgrass, and ethanol uses with the dilution proportion of 50% (v/v) in water.

In addition, the invention still further relates to the water containing ethanol extraction method of the aerial parts of grass (for example, being preferably nardgrass).

The method that obtains the nardgrass extract is used to destroy the physical operations of plant tissue from the plant extract process in the presence of organic solvent, obtain the liquid extract of thick plant extract of the aerial parts of grass family family plant.

In order to obtain extract, preferably use the leaf of plant nardgrass, be used to destroy the physical operations use turbine extraction of plant parts, the organic solvent of use can be the alcohol that comprises 2-6 carbon atom, for example ethanol, isopropyl alcohol, butanols and amylalcohol.More preferably, the physical process of selection is that turbine extracts, and organic solvent is an ethanol.Ethanol can use with different dilution factors, but preferred alcohols is used with 50,70 and 94.6% (w/w).

Taking out organic solvent by part then concentrates thick plant extract.Concentrating of extract by carrying out under 35-55 ℃ and reduce pressure (vacuum), evaporating under the controlled temperature and pressure.

Preferably, can use rotary evaporator to cause the evaporation of used organic solvent under the temperature fully.Then with the spissated extract lyophilizing of the thick plant of leaf.Stage subsequently comprises the evaluation of technological parameter and mycocidal therapeutic activity aspect.

Measure of the evaluation of nardgrass extract by measuring minimal inhibitory concentration (CIM) and minimal fungicidal concentration (CFM) to the extracorporeal antifungal activity of tinea unguium stimulus object.

Measure minimal inhibitory concentration (CIM) by the method at the Microdilution of juice, the standardization standard according to being announced in file M-27A10 by the National Committee for Clinical Laboratory Standards has some adjustment.

The plastics micro plate of sterilization (Nunclon, Delta, Nunc A/S, Roskilde tests in Denmark), comprises 96 holes to eight row form tissues of H sign with A, 12 holes of every behavior 1-12 numeral number.Every row (A-H) is corresponding to the species of a fungus, and it accepts the inoculum of the 100 μ l that measure, every row be received in YNBG with 2 times of dilutions in the juice up to 1/128 dilution nardgrass extract.The reference yeast that on each plate, comprises negative control, positive control and Candida parapsilosis (ATCC 22019).At spectrophotometer (Baush﹠amp; Lomb) regulate inoculum density in, so that, make that this suspension vol of 100ml in each hole that is added into plate comprises 0.5-2.5 * 10 corresponding to turbidity at the pipe 0.5 of the MacFarland of 530nm wavelength ratio ³UFC/ml.With plate incubation in steriliser of combination like this, carry out daily monitoring, in saccharomycetic situation 35 ℃ of incubations 48 hours, in the situation of dermatophytes ambient temperature incubation seven days.For the sufficient incubation of every kind of fungus after the time, by the reading of the test of the reflection visual comparison in mirror.

CIM regards the Cmin (Fig. 3) of the freeze-dry extract of the nardgrass that can 100% suppresses every kind of fungal isolates growth as, with reference to its corresponding positive control.In order to measure CFM, will transfer to Sabouraud glucose agar medium (Sabouraud Dextrose Agar medium) in the aliquot sample that the test that is used for measuring CIM does not present the cultivation of growth, with respect to the culture medium that does not contain medicine.The Cmin of prevention conk is regarded CFM as.

The present invention also provides the pharmaceutical composition that comprises the nardgrass water-alcohol extraction to be used to suppress the growth of dermatophytes or makes its dead purposes, described dermatomycosis fungus is trichophyton (Trichophyton spp), Microsporon (Microsporum spp) and Epidermophyton (Epidermophytom floccosun) for example, and is used to suppress by the yeast purposes of candidal disease for example.

Pharmaceutical preparation can be used as avette dose of tincture, lotion, gel, unguentum, cream, vagina and the form existence of tablet, perhaps tincture, granule, capsule and the tablet for being used to orally use that is used for topical application.

In order to understand object required for protection better, furnishing an explanation property embodiment should not be seen as the restriction to applicant right subsequently.

The example that is preferred for a kind of pharmaceutical composition of every kind of fungal disease of the present invention can comprise the nardgrass alcohol extract that is used for the saccharomycetic at least 1250 μ g/ml amount of tinea unguium; The nardgrass alcohol extract that is used at least 625 μ g/m l amount of dermatophytes; With the nardgrass alcohol extract of at least 625 μ g/ml amount that is used to suppress vaginal yeast (Candida albicans), except excipient and/or diluent, eluent and other acceptable medicinal auxiliary agent.

The acquisition of nardgrass extract

Embodiment 1.A: the collection of vegetable matter

During month of in May, 2005, collect the leaf of nardgrass, as the initiation material of the extract that is used for the present composition in the medicinal plants nursery of State University of Maring á (UEM).Plant is confirmed by the botanical analysis and by confirming with the link coupled gas chromatography of mass spectrum.The dry thing of this material is deposited in No. 11747 UEM herbariums.

Embodiment 1.B: the sign of the technical parameter of plant initiation material.

Determine the technical parameter of plant nardgrass (n=3), for example: loss on drying (PS), dehydration weightless (PD), total flavonoid (flavinoids) content (FT), total polyphenols content (PT) and volatile matter oil content (OV).The result is included in the table 1.

The numerical value (n=3) of total flavonoid (FT) that table 1. is obtained by the initiation material nardgrass, total polyphenols (PT), volatile matter oil (OV), dehydration weightless (PD) and loss on drying (PS).

Embodiment 1.C: the preparation of extract

Fresh nardgrass leaf is cleared up with compressed air, and being cut into small pieces and using with the dry extract with respect to nardgrass is 15 minutes (n=3) of the pure turbine extraction of 70% (p/p) of 20% (p/p) ratio.Extract is filtered, in rotary evaporator, concentrate lyophilizing subsequently.Before drying, determine the technical parameter in these extracts, for example dry residual (RS) and p pH, and after drying, obtain total flavonoid (FT), total polyphenols (PT) and antifungal activity.The result is included in the table 2.

Table 2. derives from the pH of the extract of nardgrass cured leaf, dry residual (RS), total flavonoid (FT) and total polyphenols (PT) (n=3).

Embodiment 2: the analysis of biological activity determination value

In order to measure the antifungal activity of nardgrass extract, use the method for detailed earlier herein for the different fungal isolates in clinical source.

Fungus was brought back to life 24/48 hour, by the inoculum of this growth-gen preparation in Sterile Saline, by Bauch ﹠amp in Sabouraud agar glucose (SDA) culture medium at 30 ℃; The Lomb spectrophotometer is regulated cell density at 530nm with the transmitance of 90+/-2%.This is cultivated and produces 1.0-5.0x10 ⁶Ufc/ml, (YeastNitrogen base glucose, YNBG) the new dilution in obtains 0.5-2.5x10 at yeast nitrogenous base glucose by its preparation ³The required final inoculum of ufc/ml.

Measure minimal inhibitory concentration inhibition concentration (CIM) by method at the Microdilution of juice, according to the standardization standard of in file M-27A, announcing by the NATIONAL COMMITTEE FOR CLINICAL LABORATORYSTANDARDS (INCCLS, 1997).

The plastics micro plate of sterilization (Nunclon, Delta, Nunc A/S, Roskilde tests in Denmark), comprises 96 holes to eight row form tissues of H sign with A, 12 holes of every behavior 1-12 numeral number.Every row (A-H) is corresponding to the species (inoculums of 100 μ l of mensuration) of a fungus, and every row are received in YNBG with the nardgrass extract of 2 times of dilutions in the juice up to 1/1024 dilution factor (corresponding to the ultimate density of 9 μ g/ml).On each plate, comprise the negative control of dilution and the reference yeast of positive control and Candida parapsilosis (ATCC 22019).

With the plate of combination thus in steriliser 35 ℃ of incubations 72 hours, carry out daily monitoring.After 72 hours, the visual comparison of being undertaken by the reflection in mirror obtains test reading.CIM regards the Cmin of the dry extract of the nardgrass that can 100% suppresses every kind of conk as, with its corresponding positive control as a reference.In order to determine the minimal fungicidal concentration (CFM) of nardgrass, the aliquot sample that derives from the CIM hole that will not observe growth shifts, and is used for comparing with the culture medium that does not contain medicine.Stop the minimum dilution of yeast growth to regard CFM as.

In order to analyze the result of minimal inhibitory concentration (CIM) and minimal fungicidal concentration (CFM), CIM and the CFM that obtains by institute's choosing method analyzed:

A) CIM of nardgrass extract and the upper and lower bound of CFM are represented in the variation of numerical value, with respect to the saccharomycetic different plant species of being tested.

B) CIM50 and CIM90 are defined as the minimal inhibitory concentration of the nardgrass extract that can suppress institute's test specimen 50% and 90% growth respectively.

C) CFM50 and CFM90 are defined as the minimal fungicidal concentration of the nardgrass extract that can prevent institute's test specimen 50% and 90% growth respectively.

The saccharomycetic effect of 2.1 pairs of tinea unguium of embodiment

Used from patient's finger first ambulatory and the isolating 19 primary yeast bacterium of toenail.The yeast causer of tinea unguium has been contained following species: Candida albicans (Candida albicans), Oidium tropicale, Candida glabrata and Candida parapsilosis.

The result shows, and is with the minimal inhibitory concentration that 10mg nardgrass extract uses the ratio of 1mg diluent, different with total fluid medium of 0.6-1.25mg/ml.The numerical value of CFM is identical with the numerical value of CIM, presents identical behavior in all species, and is as shown in table 3.

Table 3. nardgrass extract is to the variation of representing with mg/ml of various saccharomycetic minimal inhibitory concentration (CIM) and minimal fungicidal concentration (CFM).

The effect of the dermatophytes in 2.2 pairs of tinea unguium of embodiment

Fingernail and the isolating 20 kinds of dermatophytess of toenail have been used from non-sickbed patients.The fungus causer has been contained following species: Trichophyton mentagrophytes (Trichophytonmentagrophytes), trichophyton (T.tonsurans), trichophyton (T.raubitscheki), Sabouraudites lanosus (Microsporum canis), microsporon gypseum (M.gypseum), Microsporum ferrugineum (M.ferrugineum).

Result in the table 4 shows that the nardgrass extract has the antifungal potentiality to dermatophytes, and CMI numerical value is between 0.075-0.6mg/ml.Extract shows for all dermatophytess all has fungicidal action, and CMI has identical variation, all is between 0.075-0.6mg/ml.For the most responsive dermatophytes of nardgrass extract is Sabouraudites lanosus and trichophyton, is 0.075mg/ml.Trichophyton presents maximum CIM and CFM variation coefficient, is 0.075 and 0.6mg/ml.CMI for other dermatophytes does not almost change (Sabouraudites lanosus, microsporon gypseum and Trichophyton mentagrophytes) or does not change (Microsporum ferrugineum and trichophyton).

Table 4. nardgrass extract is to the variation of representing with mg/ml of the minimal inhibitory concentration (CIM) of various dermatophytess and minimal fungicidal concentration (CFM).

Embodiment 2.3 is for the saccharomycetic effect of female vulva vaginal candidiasis

Use derives from 23 kinds of vulvovaginal separators of the Candida albicans of ambulant case.

The result of table 5 shows that the minimal inhibitory concentration of nardgrass extract is 0.018-0.62mg/ml, shown in the data in the table 6.The numerical value of CFM is identical with the numerical value of CIM, all presents identical performance for all yeast.

Therefore, test variable concentrations to the work of the 19 primary yeast bacterium that derive from the tinea unguium patient, 20 kinds of dermatophytess and 23 kinds of vaginal yeasts and a kind of standard sample (ATCC) during at us in order to the CIM that obtains extract and CFM, might record nardgrass extract in the 0.018-1.25mg/ml scope can suppress the fungus of testing totally.

Table 5. nardgrass extract is to the variation of representing with mg/ml of the minimal inhibitory concentration (CIM) of the oidiomycetic vagina separator of white and minimal fungicidal concentration (CFM).

According to bioactive result, proved that the nardgrass extract provides the performance that has excellence for isolating fungus in people's clinical setting in vitro tests.And this activity is not only and is suppressed fungus, and is antifungal, even be still like this at low concentration, and this activity does not change with used extracting liq function.

The optimization that embodiment 3. extracts

Use three kinds of ethanol dilution things (50,70 and 94.6% (p/p)) to obtain extract, nardgrass concentration is between 10,20 and 30% (p/p).Extract is filtered, in rotary evaporator, concentrate lyophilizing subsequently.They are carried out the mensuration of dry residual, antibiotic evaluation, be used for optimizing and extract.For leaching process subsequently, determine following parameter: 1% pH value of aqueous solution, organoleptic feature (color, aroma and flavor), dry residual, activity substance content separate (CCD) with thin layer chromatography.

Dry residual result is included in table 6 and the table 7.

Table 6. derives from the numerical value of dry residual (RS) of the extract of the fresh leaf of nardgrass (n=12)

The numerical value that dry residual (RS) of the dry leaf of table 7. supplier and UEM nursery (n=9) obtains

Note: S=standard deviation, CV%=relative standard deviation

In order to prove best extraction, all extracts are carried out the evaluation of antifungal activity.Carry out identical experiment, difference is to use fresh and exsiccant leaf different with supplier.The result is included in the table 8.This optimization method for antifungal activity shows that the water containing ethanol extraction liquid among dilution 50,70 and 94.6% (p/p) is enough to obtain the nardgrass extract and does not interfere it for the antibacterial activity in vitro from isolating yeast of patient and standard yeast (ATCC).For preferred plant concentration, between 10-20% (p/p), obtain best response.

Table 8. nardgrass extract is to the variation of representing with mg/ml of the minimal inhibitory concentration (CIM) of yeast (Candida albicans and Oidium tropicale) and minimal fungicidal concentration (CFM).

The above-mentioned embodiment that provides is not used in technology and the method that restriction is used to obtain and prepare water-alcohol extraction of the present invention, water-alcohol extraction of the present invention is used as active antifungal, be used for pharmaceutical compositions, pharmaceutical composition can be used as liquid, gel, unguentum, cream, avette dose, capsule and tablet form exists, be used for oral and topical application, be preferably used in topical application, be used to resist tinea unguium, dermatomycosis and candidiasis.

Claims (12)

1. have the anti-fungal activity medicaments compositions, be characterised in that to comprise the nardgrass water alcohol plant extract that concentration is 5-40% (p/p) and the alcohol organic solvent of at least a C1-C10 carbon atom, be used for the treatment of tinea unguium, dermatomycosis and candidiasis.

2. the pharmaceutical composition of claim 1 is characterised in that to comprise the nardgrass plant extract that concentration is preferably 10-30% (p/p).

3. claim 1 and 2 pharmaceutical composition are characterised in that described alcohol organic solvent is alcoholic acid 50% aqueous solution, are 1: 10 ratio with respect to the dry extract of nardgrass.

4. the pharmaceutical composition of claim 1-3 is characterised in that described alcohol organic solvent is a glycol.

5. the pharmaceutical composition of claim 1-4 is characterised in that described glycol is a dipropylene glycol, is 0.5: 20 ratio with respect to the dry extract of nardgrass.

6. the pharmaceutical composition of claim 1-5 is characterised in that preferably dipropylene glycol of described alcohol organic solvent, is 10: 1 ratio with respect to the dry extract of nardgrass.

7. the pharmaceutical composition of claim 1-6, be characterised in that the fungus inhibitory action that produces 50%-100%: Sabouraudites lanosus (Mycrosporum canis) to following dermatophytes, Microsporum ferrugineum (Mycrosporum ferrugineum), microsporon gypseum (Mycrosporum gypseum), Trichophyton mentagrophytes (Trichophytum mentagrophytes), trichophyton (Trichophytum raubistscheki) and trichophyton (Trichosporum tonsurans), and yeast: Candida albicans, Candida glabrata, Candida parapsilosis and Oidium tropicale.

8. the purposes of the water-alcohol extraction of nardgrass is characterised in that to be used for pharmaceutical compositions, is used for the treatment of tinea unguium, dermatomycosis and candidiasis.

9. the purposes of claim 8, be characterised in that the fungus inhibitory action that produces 50%-100%: Sabouraudites lanosus (Mycrosporum canis) to following dermatophytes, Microsporum ferrugineum (Mycrosporum ferrugineum), microsporon gypseum (Mycrosporumgypseum), Trichophyton mentagrophytes (Trichophytum mentagrophytes), trichophyton (Trichophytum raubistscheki) and trichophyton (Trichosporumtonsurans), and yeast: Candida albicans, Candida glabrata, Candida parapsilosis and Oidium tropicale.

10. be used for the method for the water-alcohol extraction of compositions 1 from the leaf acquisition of nardgrass, be characterised in that may further comprise the steps:

A. in the presence of the alcohol organic solvent that comprises 1-6 carbon atom, destroy plant tissue;

B. by under the condition of controlled temperature and pressure, passing through the evaporation and concentration organic solvent; With

C. lyophilizing.

11. the method for claim 10 is characterised in that and uses ethanol as the alcohol organic solvent that comprises 1-6 carbon atom.

12. the method for claim 10 and 11 is characterised in that the evaporation of carrying out organic solvent under 35-55 ℃ temperature range and reducing pressure (vacuum).

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